Event Abstract

1
University of Nottingham, School of Electronic and Electrical Engineering, United Kingdom

2
University of Nottingham, School of Life Sciences, United Kingdom

Motivation
Rapid volume transmission processes such as phasic neuromodulator signalling involves changing concentrations of agonist molecules around the cells at time scales of just a few seconds(1). These processes can be studied in-vitro using microfluidic systems capable of rapidly washing agonists on and off the cells. Achieving such time scales would, however, involve rapid flow rates of hundreds of µm/sec, the functional consequences of which have yet to be characterized. In this study, we monitored the activity of hippocampal neuronal cultures grown in microfluidic devices bonded to MEAs and subjected to rapid microfluidic flow. We identified conditions in which the network dynamics of the culture are preserved under flow.
Material and Methods
Microfluidic devices were fabricated using double sided silicone tape (3M 96042) excised to required geometries and taped to Polyethylenimide coated commercial MEAs with a Polydimethylsiloxane roof. Primary hippocampal neurons (Wistar E18) were seeded into the devices at a plating density of 7000k cells/mL. The flow protocol was applied using a pressure driven flow system (OB1, Elveflow). Devices were immersed in 4 ml of culture media and a support culture (comprising 250k cells) was seeded on the glass outside the channel for conditioning purposes. This 4mL of conditioned media was used during the flow session to flow through the device. Neuronal spiking activity was recorded using a commercial 60 channel amplifier (MCS, USB-MEA60). Evoked responses were elicited by applying bi-phasic voltage pulses (700 mV) to a single electrode at a frequency of 0.2 Hz.
Results
Flow experiments were performed on 3 age groups (in days in vitro): 12–15 (“young”), 16-19 (“intermediate”) and 20-23 (“old”). Baseline spontaneous and evoked activity was recorded for 45 minutes. The device was then connected to the flow system and the same measurements were made under rapid flow (100 nL/sec; 200 µm/sec).
At baseline, the cultures exhibited synchronized bursting dynamics as expected for the given stage of development. Under flow, activity in “young” cultures switched to an asynchronous mode and spiking activity became silenced over time (figures 1A, 2A-B). In this case, stimulation specific responses vanished (figures 1C, 2C).
In “old” cultures, spiking activity and evoked responses persisted throughout the recording period but were not as stable as the control (no flow), showing approximately a 2-fold increase initially before declining to sub-baseline levels (figures 1B,D 2A,C). In this case, activity maintained synchronized dynamics similar to control but drifted slowly to less synchronized levels (figures 2B).
“Intermediate” age cultures displayed responses with mixed characteristics (figures 2A-C). Spontaneous activity was generally maintained but was much more variable than in the “old” cultures. Evoked responses vanished in some cases, and in others reversed polarity with the stimulation actually producing a pause in the ongoing activity. In this case, synchronicity levels dipped rapidly but were higher than “young” cultures.
Discussion
We showed that, using the flow protocol provided here, mature cultures at the third week in vitro preserve their activity dynamics whereas younger cultures suffer a functional disruption typically comprising a complete abolishment of spontaneous and evoked activity. This result constrains the culture developmental stage which could be studied with this approach. Nevertheless, MEA investigations are commonly performed beyond 3-4 weeks in vitro, as the cultures reach a stable functional state(2).
The deleterious effect of the flow observed here might be shear related. A possible explanation for the improved performance of mature culture could be the presence of more developed extra cellular matrix serving as a protective barrier. Thus, incorporation of shear mitigation measures in the microfluidic devices could potentially improve the performance.
Conclusion
In summary, we demonstrated a rapid microfluidic flow protocol under which neuronal cultures can preserve their activity dynamics. This result is an essential precursor to experimental models of rapid volume transmission processes such as phasic neuromodulator signalling(1) and extra-synaptic transmitter release(3).
Figure Legends
Figure 1: Activity dynamics are preserved for ”old” but not “young” cultures. (A-B) Typical raster plots of spiking activity 10 minutes before and after flow initiation (at 0 minutes). Data presented in 0.5 second bins. (C-D) Post stimulus time histograms 10 minutes before and after flow initiation. Each line shows a post-stimulus response as a cumulative raster plot from all MEA channels.
Figure 2: Measures of activity under flow and in control conditions (no flow). Individual flow curves are normalized to a baseline recording period prior to initiation of flow and then averaged to generate the shown curves. Control curves are normalized to the mean over the first 60 minutes of the recording. Shaded areas show the standard deviation. Stimulation response is computed as the difference between the number of spikes recorded during a 190 ms window starting 10 ms after the a stimulation pulse and a similar window 2000ms after the stimulation. Correlation coefficient for a given recording is the mean of the cross correlation matrix computed over smoothed raster plots from all channels. Statistics used for generating averaged curves: n=4 for “young“, n=3 for “intermediate“, n=8 for “old“, n=3 for “young“ control, n=5 for “old“ control.
References
1.Rice, M. E. & Cragg, S. J. Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway. Brain Res. Rev. 58, 303–13 (2008).
2.Chiappalone, M., Bove, M., Vato, A., Tedesco, M. & Martinoia, S. Dissociated cortical networks show spontaneously correlated activity patterns during in vitro development. Brain Res. 1093, 41–53 (2006).
3.Mann, E. O. & Mody, I. Control of hippocampal gamma oscillation frequency by tonic inhibition and excitation of interneurons. Nat. Neurosci. 13, 205–12 (2010).

Figure 1

Acknowledgements

This work was made possible thanks to the EPSRC (EP/H022112/1) and European Commission FP7 Initial Training Network NETT (289146).